本文選題：腎細胞癌 + VHL��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究目的:絕大多數(shù)腎臟惡性腫瘤的組織學(xué)類型是腎透明細胞癌。約75%-80%的腎透明細胞癌患者中存在染色體缺失、基因突變及啟動子甲基化導(dǎo)致的VHL等位基因失活表達缺失。很多研究也發(fā)現(xiàn)了VHL基因缺失、表達失活在腎透明細胞癌的發(fā)生及進展過程中發(fā)揮著非常重要的作用。氧存在時VHL蛋白(p VHL)呈遞缺氧誘導(dǎo)因子HIF-α并與之結(jié)合使其通過泛素連接酶-蛋白酶體途徑降解,使HIF-α分子在細胞中處于合成與降解的動態(tài)平衡。腎細胞癌細胞系786-O細胞中VHL基因表達缺失,細胞內(nèi)HIF-2α不能被識別并降解呈現(xiàn)持續(xù)高表達的狀態(tài),進一步誘導(dǎo)下游百余種靶基因(HRGs)的表達。課題組前期實驗中通過裸鼠體內(nèi)成瘤的實驗證實了HIF-2α對腎癌細胞系786-O成瘤的重要作用,但HIF下游靶基因?qū)δI癌細胞的生長則表現(xiàn)為促進、抑制或沒影響等不同情況。通過基因序列表達譜分析發(fā)現(xiàn)乳腺癌缺失基因-1(DBC1)及斯鈣素蛋白-1(STC1)屬于被HIF-2α誘導(dǎo)表達升高的下游靶基因,而且檢索文獻發(fā)現(xiàn)DBC1、STC1在多種惡性腫瘤均發(fā)揮著重要的作用,但它們對腎細胞癌腫瘤形成的作用及機制并沒有系統(tǒng)深入的研究。本研究的目的在于驗證DBC1、STC1會被HIF-2α誘導(dǎo)表達,屬于HIF-2α下游基因,并探討它們對腎細胞癌腫瘤形成的作用及可能機制,旨在尋找潛在的腎細胞癌靶向治療的新靶點及診斷或預(yù)后新的生物學(xué)標志物,優(yōu)化腎癌治療的個體化方案,改善晚期腎癌患者的預(yù)后。研究方法:1.培養(yǎng)本室構(gòu)建并保存的四種人類腎透明細胞癌細胞786-O(VHL-/-、VHL+/+、VHL-/-HIF-2αsh566、VHL+/+HIF-2α-d PA),使用實時熒光定量PCR技術(shù)檢測DBC1、STC1在四種細胞系中m RNA水平的表達,驗證其是否屬于HIF-2α誘導(dǎo)表達升高的基因;2.通過使用慢病毒攜帶sh RNA DBC1、sh RNA STC1及SCR感染786-O(VHL-/-)腎癌細胞分別沉默DBC1、STC1的表達及對照組細胞,嘌呤霉素篩選得到穩(wěn)定轉(zhuǎn)染的細胞系,使用Western Blot檢測相應(yīng)細胞中DBC1、STC1的表達情況;3.MTT分別檢測陰性對照的786-O(VHL-/-)細胞和沉默DBC1表達及沉默STC1表達的786-O(VHL-/-)細胞的體外增殖情況;4.分別使用沉默DBC1表達及沉默STC1表達的786-O(VHL-/-)細胞注入裸鼠一側(cè)皮下作為實驗組,對側(cè)皮下注入攜帶對應(yīng)陰性對照的786-O(VHL-/-)細胞作為對照組來進行體內(nèi)成瘤實驗,8周后處死裸鼠取出皮下腫瘤組織,進行稱重后統(tǒng)計并比較兩組實驗組及對照組裸鼠移植瘤生長情況。結(jié)果:1.DBC1及STC1在786-O(VHL+/+、VHL-/-HIF-2αsh566)細胞的表達情況低于786-O(VHL-/-、VHL+/+HIF-2α-d PA)細胞中的表達情況,表明DBC1和STC1屬于HIF-2α的下游靶基因,能夠被HIF-2α誘導(dǎo)而表達升高。2.MTT實驗結(jié)果表明沉默DBC1在786-O(VHL-/-)細胞中的表達沒有對腎癌細胞的體外增殖產(chǎn)生影響,沉默STC1在786-O(VHL-/-)細胞中的表達則抑制腎癌細胞的體外增殖。3.沉默DBC1和STC1在786-O VHL-/-細胞中的表達后裸鼠移植瘤成瘤能力明顯小于對照,DBC1組和STC1組中實驗組與對照組的差異均具有統(tǒng)計學(xué)意義(P0.01)。結(jié)論:1.DBC1、STC1在人類腎透明細胞癌細胞786-O中被HIF-2α誘導(dǎo)表達升高,是HIF-2α的下游靶基因。2.DBC1的表達對腎癌細胞體外增殖沒有影響,STC1的表達促進腎癌細胞的體外增殖;DBC1和STC1都明顯促進裸鼠移植瘤成瘤,提示它們在腎癌中是促癌因素。沉默DBC1及STC1表達抑制了786-O(VHL-/-)腎癌細胞系裸鼠移植瘤的形成,表明DBC1、STC1是可能的腎癌分子靶向治療的新靶點。3.VHL-HIF-HRGs調(diào)節(jié)通路是促進腎細胞癌發(fā)生發(fā)展的關(guān)鍵,但仍有一大批被HIF-2α誘導(dǎo)表達升高的靶基因在腎細胞癌腫瘤形成的過程中發(fā)揮的作用還沒有被系統(tǒng)得證實,它們可能通過參與不同的信號通路,發(fā)揮著對腎細胞癌發(fā)生發(fā)展相關(guān)的作用。對這些基因進行進一步的篩選和驗證有助于深入全面地了解腎細胞癌腫瘤發(fā)生發(fā)展的機制,并可以探索更多腎癌分子靶向治療的新靶點。
[Abstract]:Objective: the histologic type of the vast majority of renal malignant tumors is clear cell carcinoma of the kidney. There are chromosomal deletion, gene mutation and promoter methylation of VHL allele inactivation in about 75%-80% of renal clear cell carcinoma. Many studies have also found VHL gene deletion and inactivation in renal clear cell carcinoma. When oxygen exists, the VHL protein (P VHL) presents hypoxia inducible factor HIF- alpha and combines with the ubiquitin ligase proteasome pathway to make the HIF- alpha molecule in the cell in the dynamic equilibrium of synthesis and degradation. The expression of VHL gene in the cell line of renal cell carcinoma cell line 786-O is deficient. The HIF-2 alpha in the cell can not be identified and degraded to present a continuous high expression state, which further induces the expression of more than 100 target genes (HRGs) in the lower reaches of the group. In the early experiments of the group, the tumor formation in nude mice confirmed the important role of HIF-2 alpha on the tumor cell line 786-O in the renal cell line, but the target gene of the downstream HIF on the growth of renal cell carcinoma cells -1 (DBC1) and -1 (STC1) are the downstream target genes which are elevated by HIF-2 alpha induced expression by gene sequence expression spectrum analysis, and the retrieval literature found that DBC1, STC1 plays an important role in various malignant tumors, but they are to renal cells. The role and mechanism of cancer formation have not been systematically studied. The purpose of this study is to verify that DBC1, STC1 can be induced by HIF-2 alpha, belongs to the HIF-2 alpha downstream gene, and to explore the role and possible mechanism of them on the formation of renal cell carcinoma, aiming to find new targets and diagnosis or prognosis of potential treatment of renal cell carcinoma. A new biological marker to optimize the individualized regimen for the treatment of renal carcinoma to improve the prognosis of patients with advanced renal cancer. 1. to cultivate four kinds of human renal cell carcinoma cell 786-O (VHL-/-, VHL+/+, VHL-/-HIF-2 a sh566, VHL+/+HIF-2 alpha -d PA) constructed and preserved in this room, and detect DBC1 with real-time fluorescent quantitative PCR technique, STC1 in four kinds of cells The expression of M RNA level in the system is to verify whether it belongs to the gene of elevated HIF-2 alpha induced expression; 2. by using lentivirus to carry sh RNA DBC1, sh RNA STC1 and SCR infected 786-O (VHL-/-) kidney cancer cells are silent, and the cells of the control group and purinamycin are screened for the stable transfected cell lines. The expression of DBC1 and STC1 in the cells; 3.MTT detection of negative control 786-O (VHL-/-) cells and silent DBC1 expression and the proliferation of 786-O (VHL-/-) cells expressing STC1 expression, respectively. 4. cells were injected subcutaneously into the nude mice by silencing DBC1 expression and silent STC1 expression as experimental group, subcutaneous injection of the contralateral side 786-O (VHL-/-) cells with negative control were used as the control group to carry out the tumor formation experiment in the body. After 8 weeks, the nude mice were killed and the subcutaneous tumor tissues were taken out. After weighing, the growth of the transplanted tumor in the two groups and the control group was compared. Results: the expression of 1.DBC1 and STC1 in 786-O (VHL+/+, VHL-/-HIF-2 alpha sh566) cells was low. The expression in 786-O (VHL-/-, VHL+/+HIF-2 alpha -d PA) cells indicates that DBC1 and STC1 belong to the downstream target gene of HIF-2 a, which can be induced by HIF-2 alpha and expressed in.2.MTT. The expression of.2.MTT in.2.MTT shows that the expression of silent DBC1 in 786-O cells does not affect the proliferation of renal cell carcinoma cells. The expression of.3. inhibited the proliferation of renal cell carcinoma cells in vitro and the expression of DBC1 and STC1 in 786-O VHL-/- cells was significantly less than that of the control. The difference between the experimental group and the control group in the DBC1 and STC1 groups was statistically significant (P0.01). Conclusion: 1.DBC1, STC1 is HIF-2 alpha in the 786-O of human renal cell carcinoma cells. The expression of.2.DBC1, the downstream target gene of HIF-2 alpha, has no effect on the proliferation of renal cancer cells in vitro. The expression of STC1 promotes the proliferation of renal cancer cells in vitro, and both DBC1 and STC1 obviously promote the tumor formation in nude mice, suggesting that they are cancer promoting factors in renal carcinoma. The silence of DBC1 and STC1 inhibits the 786-O (VHL-/-) renal cell line. The formation of xenografts in nude mice shows that DBC1, STC1 is a possible new target of molecular targeting therapy for renal cancer,.3.VHL-HIF-HRGs regulation pathway is the key to promote the development of renal cell carcinoma, but a large number of target genes which are induced by HIF-2 alpha induced expression have not been systematically confirmed by the role of the target gene in the process of renal cell carcinoma. They may play a role in the development of renal cell carcinoma by participating in different signaling pathways. Further screening and verification of these genes will help to understand the mechanism of the development of renal cell carcinoma and explore more new targets for molecular targeting therapy of renal cell carcinoma.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.11

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本文編號：1959247